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PCR and real-time PCR for identification and quantification of cyanobacteria toxic species and cyanotoxin-producing genotypes in Macau main storage reservoir
Lou I.2; Xu S.2; Zhang W.2; Kong Y.1; Mok K.M.2
2014
AbstractFreshwater algal blooms have become a growing concern in Macau Storage Reservoir (MSR), which were caused by a high level of cyanobacteria, particularly Microcystis spp. and C. raciborskii that can produce microcystin and cylindrospermopsin, respectively. Long-time exposure to these cyanotoxins may affect public health, thus reliable detection and quantification of the algae species were challenging in water quality management. The aim of this study was to develop accurate and sensitive molecular methods on detecting cyanobacterial species and cyanotoxins-producing genotypes. The cyanobacteria, Microcystis spp. and C. raciborskii were identified and quantified by polymerase chain reaction (PCR), multiplex PCR and real-time quantitative PCR (qPCR) techniques. As well, the cyanotoxins-producing genotypes were also analyzed by PCR and qPCR using cylindrospermopsin polyketide synthetase gene (pks) and microcystin synthetase genes (mcys). The related water eutrophication, phytoplankton community diversity were measured accordingly. It was found out that TSI result of 65-82, indicated that MSR was categorized as a eutrophic-hypereutrophic reservoir, with the dominance of Cyanophyta in 2011, and of Chlorophyta and Bacillariophyta in 2012. The PCR (including multiplex PCR) results showed that the techniques were successful for identifying cyanobacterial species and cyanotoxins-producing genotypes in pure cultures (or plasmid), mixed cultures, and water samples in MSR. While qPCR results were proved to be applied in quantifying the cell number of cyanobacteria, Microcystis spp. and C. raciborskii, as well as the gene copy nember of microcystin- and cylindrospermopsin-producing genotypes. When the target species above 1 million cells/L, similar cell numbers estimated by microscopic counting and qPCR were obtained. Further quantification in water samples indicated that the ratio of cells number estimated by microscopy and by qPCR was 0.4- 12.9 for cyanobacteria and 0.2-3.9 for C. raciborskii. However, Microcystis spp. was not observed by manual counting, while it can be detected at low levels by qPCR, suggesting that qPCR is more sensitive and accurate than microscopic counting. On the aspect of cyanotoxins, there was a strong correspondence between the presence of the pks gene numbers and cylindrospermopsin concentrations (R2=0.95) determined by HPLC, while weak correlations were obtained between the mcys gene numbers and microcystin concentrations. Furthermore, the pks gene numbers were strongly related to Cylindrospermopsis (R2=0.88), cyanobacterial cell numbers (R2=0.96), total algae numbers (R2=0.95) and chlorophyll-a concentrations (R2=0.83), consistent with the dominant species of Cylindrospermopsis among the cyanobacteria existing in MSR. The water quality parameters NH4-N (R2=0.68) and pH (R2=0.89) were most highly correlated with the pks gene numbers.
ISBN9781634631426;9781614709251;
URLView the original
Pages79-112
Language英語
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Document TypeBook
专题University of Macau
Affiliation1.The Macao Water Supply Company Ltd.
2.Universidade de Macau
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GB/T 7714
Lou I.,Xu S.,Zhang W.,et al. PCR and real-time PCR for identification and quantification of cyanobacteria toxic species and cyanotoxin-producing genotypes in Macau main storage reservoir[M],2014.
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